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A failure to dispose of underperforming mitochondria may underlie the complex degenerative disorder known as multisystem proteinopathy. Mutations in valosin-containing protein (VCP) cause this syndrome, which affects bone, muscle, and nerves. Two papers in the March 14 Neuron online point to defects in mitochondria—at the level of ATP production and mitophagy of damaged organelles—as problems occurring in cells with faulty VCP. “We think that because cells produce less ATP, they cannot cope with energy-demanding processes,” concluded Helen Plun-Favreau of the University College London Institute of Neurology in the U.K., one of the senior authors. That metabolic vulnerability, plus a second hit from the environment or genes, could predispose energy-hungry cells like neurons to the degeneration that occurs in MSP-related conditions, including frontotemporal dementia, amyotrophic lateral sclerosis, and parkinsonism.

Faulty Engines
In one paper, Plun-Favreau and co-senior author Andrey Abramov, also at the Institute of Neurology, report that loss of VCP function interferes with ATP synthesis. First author Fernando Bartolome and colleagues used RNA silencing to knock down VCP expression in a variety of cells in culture, including the SH-SY5Y human neuroblastoma line, mouse primary cortical neurons, and primary astrocytes. In addition, they examined fibroblasts taken from three people with MSP-linked VCP mutations, and from three control donors.

Bartolome and colleagues found that the mitochondrial membrane potential was reduced in all VCP-deficient or mutant lines. That signifies weakening of the protein gradient that drives the synthesis of ATP, and the cells produced much less of this energy-rich molecule than they should. Despite this ATP deficiency, the cells gobbled up more oxygen than usual, indicating a hyperactive respiratory chain. Together, these results indicated that VCP dysfunction uncouples ATP production from mitochondrial respiration. “All the oxygen is consumed for nothing, and the ATP is not produced properly,” Plun-Favreau said.

The researchers do not know how VCP mutations uncouple the respiratory chain, or how this defect results in the myriad symptoms of MSP. However, Plun-Favreau noted that malfunctioning mitochondria like these ought to be on track for mitophagy, or cellular digestion of mitochondria. “You do not want these damaged mitochondria to accumulate in the cells, ” she said. The second Neuron paper reports that they do build up, and explains why. VCP participates in the mitophagy pathway, report the authors at St. Jude Children’s Research Hospital in Memphis, Tennessee. “Presumably, the defect we identified in VCP function accounts for the mitochondrial defect [Plun-Favreau and colleagues] document,” wrote senior author Paul Taylor in an e-mail to Alzforum.

No Disassembly
Joint first authors Nam Chul Kim and Emilie Tresse and colleagues expressed MSP-linked VCP mutations in the muscles of Drosophila, which caused degeneration and drooping wings. This phenotype reminded Taylor of flies missing parkin and PINK1, regulators of mitochondrial quality control that cause parkinsonism when mutated (see ARF related news story on Greene et al., 2003; ARF news story on Poole et al., 2008). Given the VCP fly phenotype and the fact that MSP sometimes presents as parkinsonism or PD (Kimonis et al., 2008; Spina et al., 2012), he hypothesized that VCP, PINK1, and parkin might work in the same biochemical pathway.

What might those ubiquitinated proteins be? There are probably many, Taylor said, but they identified mitofusin, an outer mitochondrial membrane GTPase, as one. As the name suggests, it joins mitochondria together. Parkin ubiquitinates mitofusin, and the researchers discovered the GTPase bound VCP as well. Wild-type VCP was required for proper degradation of ubiquitinated mitofusin, leading to mitochondrial fragmentation and mitophagy.

Mitochondrial Mayhem
How do these defects add up to MSP? Cells constantly manage their mitochondria, clearing away rotten ones, Taylor said. Faulty VCP function, he suggested, would lead to the accumulation of damaged mitochondria—just as Plun-Favreau and colleagues observed. Taylor plans to examine mitochondrial function in mouse and human tissues, while Plun-Favreau hopes to investigate mitophagy in fibroblasts from VCP carriers.

“VCP-mediated disease would thus be, at least partially, the result of a traffic jam of dysfunctional mitochondria,” Luc Dupuis of the University of Strasbourg in France, who was not part of either study team, wrote to Alzforum (see full comment below). “The two papers complement each other and provide convincing evidence that VCP mutations alter mitochondrial functions through dysfunctional recycling of abnormal mitochondria.” Many other papers have linked neurodegeneration and mitochondrial abnormalities (e.g., see ARF related news story on Duboff et al., 2012; ARF related news story on Lim et al., 2012, and Han et al., 2012; and ARF related news story on Yao et al., 2011). However, both Dupuis and Taylor cautioned that mitochondrial problems need not be central to VCP-mediated pathology.

VCP mutations alone may not cause degeneration. Even in a person with MSP, many tissues remain unaffected. Instead, researchers suggested, the failure to make sufficient ATP would make VCP-mutant cells vulnerable to an additional insult that could create the disease state. Cell types that binge on energy—as do nerves and muscle—might be most susceptible. Plun-Favreau suggested that a second hit, such as ischemia, could push the mutant cells beyond their capacity to maintain adequate ATP supplies. That second hit might be environmental, such as pesticide exposure, suggested Flint Beal, of Weill Medical College of Cornell University in New York City, in an e-mail to Alzforum (see full comment below). Or it could be genetic, said Taylor.

Taylor speculated that second-hit genetic variants might explain why MSP affects different organ systems in different people. One mutation might sensitize muscle, for example, another—nerves. “We suspect that the conspiracy between two different gene variants adds up to cause the phenotype,” he said. “VCP touches so many different systems that it could be a second partner to a lot of different diseases.” A multiple-gene theory has already been proposed for ALS (see ARF related news story). Taylor speculated that antioxidant treatment might set the mitochondria right and alleviate symptoms.—Amber Dance

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The studies by Kim and Bartolome represent two sides of the same coin and provide solid evidence of mitochondrial involvement in VCP-related diseases. Kim et al. provide a detailed, mechanistic analysis of how VCP mutations impair mitophagy. Their results show that VCP is recruited to dysfunctional mitochondria that were ubiquitinated by parkin. The mitophagy pathway delineated by these studies is that mitochondrial dysfunction leads to mitochondrial relocalization of PINK1, in turn recruiting parkin and VCP.

The Bartolome paper is more descriptive and shows that the VCP mutations, as well as VCP loss of function, lead to mitochondrial uncoupling and subsequent loss of ATP production.
How could these two studies be linked? In the picture described by Kim and collaborators, it would be quite expected that the loss of VCP, or its mutations, leads to the accumulation of dysfunctional mitochondria, which are unable to be recycled, and, thus, a global loss of mitochondrial function. VCP-mediated disease would therefore be, at least partially, the result of a traffic jam of dysfunctional mitochondria. This provides a conceptual framework to understand why VCP mutant cells lose mitochondrial potential and the capacity to synthesize ATP. In all, the two papers nicely complement each other and provide very convincing evidence that VCP mutations alter mitochondrial function through dysfunctional recycling of abnormal mitochondria.

There are several questions raised by these studies. First, what is the disease relevance of mitochondrial dysfunction towards IBM-PFD and ALS? Kim et al. show that overexpression of mutant VCP in flies leads to motor neuron and muscle phenotypes, as well as mitochondrial abnormalities, but it remains unknown whether the mitochondrial phenotype is indeed the cause of the neurodegenerative and myopathic phenotype. As stated by the authors in their discussion, the "mitophagy-related" function of VCP is not the only VCP function altered by mutations. The critical function(s) altered by the mutations that cause the phenotype still need to be defined. Second, these papers add to the puzzling diversity of neurodegenerative diseases involving the mitochondrial quality control pathway. Despite the fact that PINK1, parkin, mitofusins, OPA1, DRP1, and now VCP are all involved to some extent in the mitochondrial quality pathway, mutations in these genes lead to a vast array of previously unlinked clinical phenotypes, from Parkinson's disease to hereditary neuropathies, and from ALS and FTD to IBM and myopathies. Dissecting how a seemingly ubiquitous mitochondrial defect leads to a cell-type selective degeneration remains a challenge. Conversely, similar VCP mutations are able to lead to completely different clinical phenotypes. Whether a second hit, either genetic or environmental, might lead VCP mutations to trigger IBM-PFD rather than ALS remains to be determined.

The AAA ATPase VCP serves as a vital nanomachine by regulating ubiquitinated substrates for proteasomal degradation. With the discovery of VCP missense mutations that link to inherited disorders such as IBM-PFD, ALS, and FTD, along with the pathological appearance of this protein in other neurological disorders, it seems that this evolutionary conserved protein may be an important clue in solving a possible convergent pathogenic mechanism among many diseases.

In the Neuron papers, the authors report that control of mitochondrial integrity has a lot to do with VCP. Dr. Taylor’s group used both genetic and cell-based analyses to show that VCP may be involved in monogenic PD pathogenesis through the degradation of the mitochondrial membrane protein mitofusin. Earlier reports using fly PD models have demonstrated that two familiar PD factors, PINK1 and parkin, can directly modulate mitochondrial dynamics. Others also revealed that VCP might do the same. Another important thing to note: This study nicely showed that sequential recruitment of parkin, followed by VCP, to the damaged mitochondria may be important for keeping these energy factories functioning properly. Although how this mechanism plays out remains to be answered, these data certainly fit into the notion that mitochondria dysfunction may be a key to PD pathogenesis.

The second paper, reported by Bartolome et al., showed that either knocking down VCP or expressing IBM-PFD-linked disease mutants could induce mitochondrial uncoupling, which resulted in reduced ATP levels. Part of their results are consistent with our earlier report showing that flies expressing pathogenic VCP alleles have reduced cellular ATP. However, given that a number of reports demonstrated VCP disease mutations could have normal or higher-than-normal ATPase activity, it is puzzling how the knockdown of this ATPase would impair mitochondrial function similarly to those disease alleles found in patients. Nevertheless, this interesting finding certainly opens a new window to further delineate VCP-associated energy metabolism. Overall, both studies attest to the idea that balanced energy production and expenditure in postmitotic cells, such as neurons, are truly essential for maintaining a healthy brain.

The link between these two papers is that VCP mutations have deleterious effects on mitochondria. However, the two groups approach this from different directions. Plun-Favreau et al. studied mitochondrial function in fibroblasts and show that there is uncoupling, which, as expected, reduces membrane potential and ATP production, but increases O2 consumption. This would be expected to make neurons and other cells more vulnerable to other insults having effects on bioenergetics such as ischemia or pesticide exposure. The second paper by Taylor et al. provides strong evidence for an important role of VCP in mitophagy. It shows that VCP can complement PINK1 deficiency but not parkin deficiency, since parkin-mediated ubiquitination is required to recruit VCP to damaged mitochondria and for degradation of mitofusins.

There were prior data showing links to mitophagy and abnormal mitochondria in mouse VCP knock-in models, but these data are much more definitive, particularly the studies of Taylor and colleagues, which provide strong genetic evidence. How do we relate this to brain, spinal cord, and muscle? That is not too hard, since both neurons and muscle cells are postmitotic and consume large amounts of energy.